Dendritic spines are actin-rich compartments that protrude from the microtubule-rich dendritic shafts of principal neurons. Spines contain receptors and postsynaptic machinery for receiving the majority of glutamatergic inputs. Recent studies have shown that microtubules polymerize from dendritic shafts into spines and that signaling through synaptic NMDA receptors regulates this process. However, the mechanisms regulating microtubule dynamics in dendrites and spines remain unclear. Here we show that in hippocampal neurons from male and female mice, the majority of microtubules enter spines from highly localized sites at the base of spines. These entries occur in response to synapse-specific calcium transients that promote microtubule entry into active spines. We further document that spine calcium transients promote local actin polymerization, and that F-actin is both necessary and sufficient for microtubule entry. Finally, we show that drebrin, a protein known to mediate interactions between F-actin and microtubules, acts as a positive regulator of microtubule entry into spines. Together these results establish for the first time the essential mechanisms regulating microtubule entry into spines and contribute importantly to our understanding of the role of microtubules in synaptic function and plasticity.

Endocytic protein trafficking is directed by sorting signals on cargo molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during endocytosis remain poorly defined. Using Caenorhabditis elegans, we demonstrate that multiple plasma membrane endocytic adaptors function redundantly to regulate clathrin-mediated endocytosis and to recruit components of the endosomal sorting complex required for transport (ESCRT) machinery to the cell surface to direct the sorting of ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the endolysosomal system. In the absence of a heterooligomeric adaptor complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of its clathrin-mediated internalization. Consistent with a role for the ESCRT machinery during cargo endocytosis, we further show that the ESCRT-0 complex accumulates at a subset of clathrin-coated pits on the surface of human cells. Our findings suggest a unique mechanism by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway.clathrin | multivesicular endosome

Summary Neurite formation is a seminal event in the early development of neurons. However, little is known about the mechanisms by which neurons form neurites. F-BAR proteins function in sensing and inducing membrane curvature [1, 2]. Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family, regulates endocytosis in a variety of cell types [3–9]. However, there is little data on how CIP4 functions in neurons [10, 11]. Here we show that CIP4 plays a novel role in neuronal development by inhibiting neurite formation. Remarkably, CIP4 exerts this effect not through endocytosis, but by producing lamellipodial protrusions. In primary cortical neurons CIP4 is concentrated specifically at the tips of extending lamellipodia and filopodia, instead of endosomes as in other cell types. Overexpression of CIP4 results in lamellipodial protrusions around the cell body, subsequently delaying neurite formation and enlarging growth cones. These effects depend on the F-BAR and SH3 domains of CIP4 and on its ability to multimerize. Conversely, cortical neurons from CIP4-null mice initiate neurites twice as fast as controls. This is the first study to demonstrate that an F-BAR protein functions differently in neuronal vs. non-neuronal cells and induces lamellipodial protrusions instead of invaginations or filopodia-like structures.

SummaryCdc42-interacting protein 4 (CIP4), a member of the F-BAR family of proteins, plays important roles in a variety of cellular events by regulating both membrane and actin dynamics. In many cell types, CIP4 functions in vesicle formation, endocytosis and membrane tubulation. However, recent data indicate that CIP4 is also involved in protrusion in some cell types, including cancer cells (lamellipodia and invadopodia) and neurons (ribbed lamellipodia and veils). In neurons, CIP4 localizes specifically to extending protrusions and functions to limit neurite outgrowth early in development. The mechanism by which CIP4 localizes to the protruding edge membrane and induces lamellipodial/veil protrusion and actin rib formation is not known. Here, we show that CIP4 localization to the protruding edge of neurons is dependent on both the phospholipid content of the plasma membrane and the underlying organization of actin filaments. Inhibiting phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) production decreases CIP4 at the membrane. CIP4 localization to the protruding edge is also dependent on Rac1/WAVE1, rather than Cdc42/N-WASP. Capping actin filaments with low concentrations of cytochalasin D or by overexpressing capping protein dramatically decreases CIP4 at the protruding edge, whereas inactivating Arp2/3 drives CIP4 to the protruding edge. We also demonstrate that CIP4 dynamically colocalizes with Ena/VASP and DAAM1, two proteins known to induce unbranched actin filament arrays and play important roles in neuronal development. Together, this is the first study to show that the localization of an F-BAR protein depends on both actin filament architecture and phospholipids at the protruding edge of developing neurons.

A number of studies have demonstrated interplay between the cytoskeleton and G protein signaling. Many of these studies have determined a specific interaction between tubulin, the building block of microtubules, and G proteins. The alpha subunits of some heterotrimeric G proteins, including Gsalpha, have been shown to interact strongly with tubulin. Binding of Galpha to tubulin results in increased dynamicity of microtubules due to activation of GTPase of tubulin. Tubulin also activates Gsalpha via a direct transfer of GTP between these molecules. Structural insight into the interaction between tubulin and Gsalpha was required, and was determined, in this report, through biochemical and molecular docking techniques. Solid phase peptide arrays suggested that a portion of the amino terminus, alpha2-beta4 (the region between switch II and switch III) and alpha3-beta5 (just distal to the switch III region) domains of Gsalpha are important for interaction with tubulin. Molecular docking studies revealed the best-fit models based on the biochemical data, showing an interface between the two molecules that includes the adenylyl cyclase/Gbetagamma interaction regions of Gsalpha and the exchangeable nucleotide-binding site of tubulin. These structural models explain the ability of tubulin to facilitate GTP exchange on Galpha and the ability of Galpha to activate tubulin GTPase.

A large percentage of current drugs target G-protein-coupled receptors, which couple to well-known signaling pathways involving cAMP or calcium. G-proteins themselves may subserve a second messenger function. Here, we review the role of tubulin and microtubules in directly mediating effects of heterotrimeric G-proteins on neuronal outgrowth, shape and differentiation. G-protein-tubulin interactions appear to be regulated by neurotransmitter activity, and, in turn, regulate the location of Gα in membrane microdomains (such as lipid rafts) or the cytosol. Tubulin binds with nanomolar affinity to Gsα, Giα1 and Gqα (but not other Gα subunits) as well as Gβ₁γ₂ subunits. Gα subunits destabilize microtubules by stimulating tubulin’s GTPase, while Gβγ subunits promote microtubule stability. The same region on Gsα that binds adenylyl cyclase and Gβγ also interacts with tubulin, suggesting that cytoskeletal proteins are novel Gα effectors. Additionally, intracellular Giα-GDP, in concert with other GTPase proteins and Gβγ, regulates the position of the mitotic spindle in mitosis. Thus, G-protein activation modulates cell growth and differentiation by directly altering microtubule stability. Further studies are needed to fully establish a structural mechanism of this interaction and its role in synaptic plasticity.

The heterotrimeric, G protein-coupled receptor-associated G protein, G␣ s , binds tubulin with nanomolar affinity and disrupts microtubules in cells and in vitro. Here we determine that the activated form of G␣ s binds tubulin with a K D of 100 nM, stimulates tubulin GTPase, and promotes microtubule dynamic instability. Moreover, the data reveal that the ␣3-␤5 region of G␣ s is a functionally important motif in the G␣ s -mediated microtubule destabilization. Indeed, peptides corresponding to that region of G␣ s mimic G␣ s protein in activating tubulin GTPase and increase microtubule dynamic instability. We have identified specific mutations in peptides or proteins that interfere with this process. The data allow for a model of the G␣ s /tubulin interface in which G␣ s binds to the microtubule plus-end and activates the intrinsic tubulin GTPase. This model illuminates both the role of tubulin as an "effector" (e.g. adenylyl cyclase) for G␣ s and the role of G␣ s as a GTPase activator for tubulin. Given the ability of G␣ s to translocate intracellularly in response to agonist activation, G␣ s may play a role in hormone-or neurotransmitter-induced regulation of cellular morphology.

The Wnt/β-catenin signaling pathway plays a key role in the progression of human colorectal cancers (CRCs) and is one of the leading targets of chemotherapy agents developed for CRC. The present study aimed to investigate the anti-cancer effects and molecular mechanisms of 19-O-triphenylmethyl andrographolide (RS-PP-050), an andrographolide analogue and determine its activity in the Wnt/β-catenin pathway. RS-PP-050 was found to potently inhibit the proliferation and survival of HT-29 CRC cells. It induces cell cycle arrest and promotes apoptotic cell death which was associated with the activation of PARP-1 and p53. Furthermore, RS-PP-050 exerts inhibitory effects on β-catenin transcription by suppressing T-cell factor/lymphocyte enhancer factor (TCF/LEF) activity in cells overexpressing β-catenin and by down-regulating the endogenous expression of Wnt target genes. RS-PP-050 also decreased the protein expression of the active form of β-catenin but functions independently of GSK-3β, a negative regulator of Wnt. Interestingly, RS-PP-050 extensively blocks phosphorylation at Ser675 of β-catenin which links to interference of the nuclear translocation of β-catenin and might contribute to Wnt inactivation. Collectively, our findings reveal the underlying anti-cancer mechanism of an andrographolide analogue and provide useful insight for exploiting a newly chemotherapeutic agent in Wnt/β-catenin-overexpressing CRC cells.